Planta 146, 643~48 (1979)
P l ~ J n ~ 9 by Springer-Verlag 1979
Soluble and Microsomal Glutathione S-Transferase Activities in Pea Seedlings (Pisum sativum L.) H. Diesperger and H. Sandermann, Jr. Institut ffir Biologie II, Biochemieder Pflanzen, Universitfit Freiburg, Sch/inzlestral3e 1, D-7800 Freiburg i.Br., Federal Republic of Germany
Abstract. Epicotyl and primary leaves of pea seedlings (Pisum sativum L., var. Alaska) were found to contain soluble and microsomal enzymes catalyzing the addition of glutathione to the olcfinic double bond o f cinnamic acid. Glutathione S-cinnamoyl transfer was also obtained with enzyme preparations from potato slices and cell suspension cultures of parsley and soybean. The pea transferases had p H - o p t i m a between p H 7 . 4 and 7.8 Km-values were 0.1 0 . 4 r a M and 1-4 m M for cinnamic acid and glutathione, respectively. V-values were between 2-15 nmol m g - 1 protein x rain. C h r o m a t o g r a p h y on Sephacryl S-200 indicated that the soluble pea glutathione S-cinnamoyl transferase activity existed in molecular weight forms of 37,000, 75,000, and 150,000. The glutathione-dependent cleavage of the herbicide fluorodifen was catalyzed by a different soluble enzyme activity which eluted in molecular weight positions of 47,000 and/or 82,000. The microsomal fraction from pea primary leaves also catalyzed the conjugation of the carcinogen benzo[e]pyrene with glutathione. Key words: Cinnamic acid - Fluorodifen - Glutathione - S-transferases Pisum. Introduction Glutathione (GSH) S-transferase activities constitute an important group of the detoxifying enzyme systems of m a m m a l i a n liver (Boyland and Chasseaud, 1969; Jakoby, 1978). Several transferase activities for the conjugation of herbicides with G S H have also been discovered in plants (Shimabukuro et al., 1978). Abbreviations: GSH = gtutathione ; DDE = 1,1-Dichloro-2,2-bis-(4chlorophenyl)-ethylene; DDMU = 1-Chloro-2,2-bis-(4-chlorophenyl)-ethylene
In experiments to trap an electrophilic intermediate of the 4-hydroxylation of cinnamic acid (Sandermann etal,, 1977; Diesperger and Sandermann, 1978), the microsomai fraction from parsley cell suspension cultures was found to catalyze the addition of G S H to the olefinic double bond of cinnamic acid (Fig. 1 A). In contrast to these results, the liver G S H S-transferases occur only as soluble enzymes (Boyland and Chasseaud, 1969; Jakoby, 1978) which are unable to add G S H to cinnamic acid (Boyland and Chasseaud, 1967). The present communication deals with the occurrence of soluble and microsomal G S H S-cinnamoyl transferase activities in pea seedlings. A comparison of the fluorodifen-cleaving enzyme of pea seedlings (Frear and Swanson, 1973; Fig. 1B) and the formation of a GSH-conjugate of the carcinogen benzo[@ pyrene is also reported.
Materials and Methods Materials
Seeds of Pisum sativum L., var. Alaska, were kindly donated by Prof. R. Hertel of this Department. [3-14C]Cinnamic acid and [7, 10-14C]benzo[c~]pyrene were purchased from CEA, Gif-surYvette, France, and Amersham-Buchler, Braunschweig, respectively. [t4C-CF3]Fluorodifen was kindly donated by Ciba-Geigy A.G., Basel. [3-14C]p-Coumaricacid was prepared from [3-14C]cinnamic acid using a parsley microsomal cinnamic acid 4-hydroxylase preparation (Diesperger et al., 1974). [3-~4C]Dihydrocinnamicacid was purchased from ICN Company, Irvine California. Soybean cell suspension cultures were grown as previously described (v.d. Trenck and Sandermann, 1978). Aged potato slices were prepared as described (Rich and Lamb, 1977). General Procedures. The amounts of radioactivity and of protein were determined according to (Diesperger et al., 1974, Diesperger and Sandennann, 1978) and (Schaffner and Weissmann, 1973), respectively. Solvent systemsfor descending paper chromatography (Diesperger and Sandermann, 1978) were; (A) butanol-l/acetic acid/water, 2/i/l, by vol; and (B) benzene/acetic acid/water, 2/2/1,
0032-0935/79/0146/0643/$ 01.20
644
H. Diesperger and H. Sandermann, Jr. : Glutathione S-Transferase Activities in Pea
Assay for GSH-Conjugation of Benzo[c~]pyrene. Microsomal sus-
by vol, upper phase. The solvent system used for thin-layer chromatography on silica gel G (Frear and Swanson, 1973) was; (C) butanol-l/acetic acid/water, 12/3/5, by vol.
pension (50pl; containing 200-300pg protein), 0.2mM [7, 10-[14C]benzo[e]pyrene (50 pl; 1.9.103 d s- 1; dispersed in 0.3% (v/v) Triton X-100, 0.3% (v/v) ethyleneglycolmonomethylether), 125 mM GSH (5 gl).
Enzyme Preparation. Pea seeds were soaked overnight in tap water and were then germinated in the dark as described by Russell (1971). After 12 days, primary leaves and 10 cm epicotyl sections were collected separately and homogenized at 4 ~ C in 1 volume (per weight) of 50 mM Tricine x KOH, pH 7.5, by means of a household mixer (Krups model 3 Mix 3,000). The supernatant of a preliminary centrifugation (20 min, 10,000 g) was subjected to ultracentrifugation (90 min, 100,000g). The microsomal pellets were suspended in small volumes of 50 mM TricinexKOH, pH 7.5, and could be stored for several weeks at - 2 0 ~ C without loss of GSH S-cinnamoyl transferase activity. The supernatants from ultracentrifugation could also be stored for several weeks at - 2 0 ~ C. They were concentrated by ultrafiltration in an Amicon cell (UM-10 filter, 3.4.101 kN m -z, 5~ C) in order to remove most of an endogeneous transferase inhibitor. In most experiments (all of the kinetic experiments reported), the soluble enzymes were further purified by chromatography on a column of Sephacryl S200 (cf. Fig. 4).
Results and Discussion
Nature of Products Formed The GSH S-cinnamoyl adducts formed by the soluble and microsomal fractions of pea epicotyl and primary leaves (see below) were isolated by paper chromatography in solvent system (A). The previous chemical methods (Diesperger and Sandermann, 1978), in particular the release of dihydrocinnamic acid upon desulfurization by Raney nickel, indicated that in all cases GSH was linked to cinnamic acid by addition to the olefinic double bond, as shown in Fig. 1 A. The product formed from the herbicide fluorodifen depended on the pH-value of the incubation mixture (Fig. 2). At pH 9.0; a product with Rf-values of 0.5 [solvent system (A)] and 0.33 [solvent system (C)] was formed. This product was probably identical with the intact GSH-adduct of Figure 1 B (cf. Frear and Swanson, 1973). However, a faster migrating product with Rf-values of 0.7 [solvent system (A)] and 0.6 [solvent system (C)] was formed at pH 7.5. At an intermediate pH-value of 8.25, both products were detected (Fig. 2). These results appeared to indicate that the soluble enzyme fraction from pea seedlings contained some peptidase for a cleavage of the intact GSH-adduct of Fig. 1 B at pH-values below 9.0. No cleavage of the GSH S-cinnamoyl adduct of Fig. 1A could be observed in the same soluble enzyme fractions at pH 7.5.
Enzyme Assays General Procedure. All solutions used to determine enzyme activities contained 50 mM Tricine x KOH, pH 7.5. The standard incubations were carried out for 60 min at 30~ C and were terminated by the application of the incubation mixture to chromatography paper, followed by development in the solvent system (A). Control incubations with heat-denatured enzyme (5 rain, 95 ~ C) were always run in parallel, and the control values of radioactivity were subtracted from the values obtained with the non-denatured enzyme. The assay mixtures were prepared by mixing the following ingredients.
GSH S-Cinnamoyl TransferaseAssay. Enzyme solution (50 gl; containing 100 300 gg protein), 1.2 mM [3-14C]cinnamic acid (50 pl; 3.8.103 d s- 1), 50 mM Tricine x KOH (20 gl) or, where indicated, 6 mM NADPH (20 pl), 125 mM GSH (5 pl) or, where indicated, 75 mM 2-mercaptoethanol (5 gl). Assay for Cleavage of Fluorodifen (cf. Frear and Swanson, 1973). Enzyme solution (50 gl; containing appr. 100 pg protein), 176 mM [14C-CF3]fluorodifen (1 pl; 7.6.102 d s- 1), 6 mM GSH (50 pl).
{•
~OOH
COOH
GSH
CINNAMIC-ACI D
~)
GSH-ADDUCT
NO2 +
FLUORODINFE
GBH-ADDUC T
o@No2
Fig. 1A and B. Glutathione-dependent reactions catalyzed by enzymes from pea seedlings. A Conjugation with cinnamic acid. The chemical characterization of the GSH S-cinnamoyl adduct did not differentiate between a thioether linkage to either C2 or C3 of cinnamic acid. B Cleavage of the herbicide, fluorodifen (Frear and Swanson, 1973)
H. Diesperger and H. Sandermann, Jr. : Glutathione S-TransferaseActivities in Pea
645
71 Q
ORG IN I
A
I
FRONT
V L
aSH
I
I
Fig. 2. Reaction between [14 C-CFa]fluorodifen and glutathione as catalyzed by the purified soluble enzyme fraction from pea primary leaves. The figure shows the distributions of radioactivity on paper chromatograms [solvent system
?
0
A/
FRONT
(A)] of incubations performed at 1: pH 9.0, 2: pH 8.25 and 3: pH 7.5. Abbreviations: GSH = glutathione; F = fluorodifen
I
(]SH 1
o
i
....
1'o
I
2'o
F I
3'o
Previous studies with whole peanut plants have indicated that the intact GSH-adduct of Fig. 1 B was processed to the S-cysteinyl adduct 1 (Shimabukuro et al., 1978).
Kinetic Properties of the GSH S-Cinnamoyl Transferase Activities from Pea Seedlings The partially purified soluble enzymes and the microsomal activities had distinct pH-optima between pH 7.4 and 7.8, whereas, the non-enzymatic conjugation of GSH and cinnamic acid (cf. Diesperger and Sandermann, 1978; about 50 pmol adduct/min in the standard assay at pH 7.5) increased with increasing pH-values. The various transferase activities were linearly dependent on the amount of protein used (at least up to 300 gg protein per assay), as well as on the incubation time (up to 60 min). The purified, soluThe second split product of fluorodifen cleavage, p-nitrophenol (cf. Fig. 1 B), is further processed in peanut to the 6-O-malonyl-/L D-glucopyranosyl-derivative (Frear, 1976). A cell-free malonyltransfer from malonyl-SCoA to/LD-glucopyranosyl-p-nitrophenolate has recently been demonstrated with a highly purified malonyltransferase from parsley cell suspension cultures (U. Matern and
H. Sandermann, unpublished results)
,
40 CM
----
ble and microsomal transferase activities were abolished by heat-denaturation (5 min, 95 ~ C). In the case of the crude soluble extract from primary leaves, heating for 2 min at 95 ~ C resulted in a 2 8-fold stimulation, and transferase activity was only abolished after 20 min at 95 ~ C. Michaelis constants were determined by the Lineweaver-Burk procedure and were, in all cases, between 0.1-0.4 m M for cinnamic acid (in the presence of 5 m M GSH) and between 1-4 m M for GSH (in the presence of 0.5 m M cinnamic acid). V was 10 15nmol adduct/mg p r o t e i n x m i n for the purified soluble enzymes and 2 - 4 n m o l adduct/ mg protein x rain for the microsomal fractions. The microsomal transferase activity remained associated with the microsomal fraction after repeated washing and after centrifugation through a 5 cm cushion of 1 M glucose, 100 mM Tris-HC1, pH 7.5 (90 min, 100,000 g).
GSH S-Cinnamoyl Transfer in Other Plants The microsomal fraction from parsley cell suspension cultures (Diesperger and Sandermann, 1978) had V, 1 nmol adduct/mg protein x min. A microsomal frac-
646
H. Diesperger and H. Sandermann, Jr. : Gtutathione S-Transferase Activities in Pea
tion from potato slices (Rich and Lamb, 1977) had V, about 10 pmol adduct/mg protein • min. When tested in the presence of 1 mM NADPH, the potato microsomal fraction formed the Rf-0.4 GSH-adduct of cinnamic acid [solvent system (A)] which was probably derived from cinnamic acid 3.4-epoxide (Diesperger and Sandermann, 1978). In addition, smaller amounts of products which co-migrated with p-coumaric and caffeic acid [solvent systems (B)] and a cinnamoylcompound remaining at the origin in the solvent system (B), were formed. These various products were not formed by heat-denatured enzyme (5 rain, 95 ~ C) and required the presence of NADPH. Addition of the soluble protein fraction of potato slices led to the previously reported increase in p-coumaric acid formation (Rich and Lamb, 1977) at the expense of the cinnamoyl-compound at the origin.
No GSH S-cinnamoyl transferase activity was detected in the crude soluble enzyme fraction from potato slices (Rich and Lamb, 1977) or from parsley cell suspension cultures (Diesperger and Sandermann, 1978). It has not been determined whether this was caused by the presence of an endogeneous inhibitor which was present in the crude soluble extract from the pea seedlings (data not shown). Soybean cell suspension cultures were found to contain soluble and microsomal GSH S-cinnamoyl transferase activities with V, 2nmol adduct/mg proteinxmin for the microsomal fraction, and V, 4.5 nmol adduct/mg protein x min for the crude soluble fraction. Intact log-phase soybean cell suspension cultures (40 ml) were incubated for 2 h at 25 ~ C with [3-~4C]cinnamic acid (0.5 mM; 3.8.10 4 d s- 1) without the addition ofGSH. A 6% conversion to the GSH S-cinnamoyl adduct of Fig. 1A was found Upon paper chromatography in the solvent system (A), and about equal amounts of the adduct were isolated from the cells and from the growth medium. A 1.4% conversion was found in the autoclaved control cultures (cf. v. d. Trenck and Sandermann, 1978).
Alternative Substrates No detailed studies on alternative transferase substrates have been carried out. This section is, therefore, limited to some qualitative results obtained with the pea microsomal fractions. A GSH-adduct was formed when [3-14C]p-coumaric acid was employed instead of [3-14C]cinnamic acid, but [3-14C]dihydro cinnamic acid did not serve as a substrate. 2-Mercaptoethanol was able to partially replace GSH, and the adduct formed with cinnamic acid had an Rf-value of 0.25-0.4 in the solvent system (B). p-Coumaric acid has the same Rf-value in this solvent system which is in use for the assay of microsomal cinnamic acid 4-hydroxylase activity (Russell, 1971). Further-
more, 2-mercaptoethanol has been reported to be a strong activator of the pea cinnamic acid 4-hydroxylase (Russell, 1971). The present results indicate that the simple addition of 2-mercaptoethanol to cinnamic acid is also catalyzed by the pea microsomal fraction, and that this reaction may interfere with the chromatographic determination of p-coumaric acid. The microsomal fraction from pea primary leaves also catalyzed the formation of a GSH-adduct with the carcinogen benzo[~]pyrene, leading to a product with an Rf-value of 0.55 in the solvent system (A) [V, 30 pmol adduct/mg protein x h). This product was not formed in the absence of GSH or with heatdenatured enzyme (5 min, 95 ~ C). Subsequent results indicated that the microsomal fraction from pea primary leaves also catalyzes the formation of oxygenated metabolites of benzoMpyrene so that the GSHadduct may be formed as a secondary product (T. v. d. Trenck and H. Sandermann, unpublished resuits).
Inhibitors The following compounds gave less than 15% inhibition of microsomal GSH S-cinnamoyl transfer when added at 5 mM to the standard assay mixture; allyl alcohol, propachlor, styrene oxide, aldrin, dieldrin, endrin, DDE, DDMU, heptachlor, heptachlorepoxide, dihydrocinnamic acid, and atrazine. These inhibition effects were corrected for the appr. 25% inhibition given by the organic solvents used at final concentrations of 4%, v/v (methanol, dimethylsulfoxide or ethyleneglycol monomethylether). The addition of 5 mM styrene resulted in 27% inhibition; 5 mM cinnamyl alcohol inhibited by 76%, and 5 mM 3.4methylenedioxy-cinnamic acid gave complete inhibition. 2-Mercaptoethanol, which was accepted as an alternative substrate (see above), was a strong inhibitor of the soluble and microsomal GSH S-cinnamoyl transfer reactions (50% inhibition at 0 . 5 - 2 mM 2mercaptoethanol). Lineweaver-Burk plots (not shown) indicated a non-competitive type of inhibition.
Apparent Molecular Weights The soluble enzyme fractions were chromatographed on a column of Sephacryl S-200, and similar results were obtained for the extracts from epicotyl and primary leaves. In the experiment shown in Fig. 3, the soluble GSH S-cinnamoyl transferase activity appeared in two molecular weight regions corresponding to 37,000 and 150,000, respectively. In other experiments an additional peak corresponding to 75,000
H. Diesperger and H. Sandermann, Jr.: Glutathione S-Transferase Activities in Pea
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P l ~ J n ~ 9 by Springer-Verlag 1979
Soluble and Microsomal Glutathione S-Transferase Activities in Pea Seedlings (Pisum sativum L.) H. Diesperger and H. Sandermann, Jr. Institut ffir Biologie II, Biochemieder Pflanzen, Universitfit Freiburg, Sch/inzlestral3e 1, D-7800 Freiburg i.Br., Federal Republic of Germany
Abstract. Epicotyl and primary leaves of pea seedlings (Pisum sativum L., var. Alaska) were found to contain soluble and microsomal enzymes catalyzing the addition of glutathione to the olcfinic double bond o f cinnamic acid. Glutathione S-cinnamoyl transfer was also obtained with enzyme preparations from potato slices and cell suspension cultures of parsley and soybean. The pea transferases had p H - o p t i m a between p H 7 . 4 and 7.8 Km-values were 0.1 0 . 4 r a M and 1-4 m M for cinnamic acid and glutathione, respectively. V-values were between 2-15 nmol m g - 1 protein x rain. C h r o m a t o g r a p h y on Sephacryl S-200 indicated that the soluble pea glutathione S-cinnamoyl transferase activity existed in molecular weight forms of 37,000, 75,000, and 150,000. The glutathione-dependent cleavage of the herbicide fluorodifen was catalyzed by a different soluble enzyme activity which eluted in molecular weight positions of 47,000 and/or 82,000. The microsomal fraction from pea primary leaves also catalyzed the conjugation of the carcinogen benzo[e]pyrene with glutathione. Key words: Cinnamic acid - Fluorodifen - Glutathione - S-transferases Pisum. Introduction Glutathione (GSH) S-transferase activities constitute an important group of the detoxifying enzyme systems of m a m m a l i a n liver (Boyland and Chasseaud, 1969; Jakoby, 1978). Several transferase activities for the conjugation of herbicides with G S H have also been discovered in plants (Shimabukuro et al., 1978). Abbreviations: GSH = gtutathione ; DDE = 1,1-Dichloro-2,2-bis-(4chlorophenyl)-ethylene; DDMU = 1-Chloro-2,2-bis-(4-chlorophenyl)-ethylene
In experiments to trap an electrophilic intermediate of the 4-hydroxylation of cinnamic acid (Sandermann etal,, 1977; Diesperger and Sandermann, 1978), the microsomai fraction from parsley cell suspension cultures was found to catalyze the addition of G S H to the olefinic double bond of cinnamic acid (Fig. 1 A). In contrast to these results, the liver G S H S-transferases occur only as soluble enzymes (Boyland and Chasseaud, 1969; Jakoby, 1978) which are unable to add G S H to cinnamic acid (Boyland and Chasseaud, 1967). The present communication deals with the occurrence of soluble and microsomal G S H S-cinnamoyl transferase activities in pea seedlings. A comparison of the fluorodifen-cleaving enzyme of pea seedlings (Frear and Swanson, 1973; Fig. 1B) and the formation of a GSH-conjugate of the carcinogen benzo[@ pyrene is also reported.
Materials and Methods Materials
Seeds of Pisum sativum L., var. Alaska, were kindly donated by Prof. R. Hertel of this Department. [3-14C]Cinnamic acid and [7, 10-14C]benzo[c~]pyrene were purchased from CEA, Gif-surYvette, France, and Amersham-Buchler, Braunschweig, respectively. [t4C-CF3]Fluorodifen was kindly donated by Ciba-Geigy A.G., Basel. [3-14C]p-Coumaricacid was prepared from [3-14C]cinnamic acid using a parsley microsomal cinnamic acid 4-hydroxylase preparation (Diesperger et al., 1974). [3-~4C]Dihydrocinnamicacid was purchased from ICN Company, Irvine California. Soybean cell suspension cultures were grown as previously described (v.d. Trenck and Sandermann, 1978). Aged potato slices were prepared as described (Rich and Lamb, 1977). General Procedures. The amounts of radioactivity and of protein were determined according to (Diesperger et al., 1974, Diesperger and Sandennann, 1978) and (Schaffner and Weissmann, 1973), respectively. Solvent systemsfor descending paper chromatography (Diesperger and Sandermann, 1978) were; (A) butanol-l/acetic acid/water, 2/i/l, by vol; and (B) benzene/acetic acid/water, 2/2/1,
0032-0935/79/0146/0643/$ 01.20
644
H. Diesperger and H. Sandermann, Jr. : Glutathione S-Transferase Activities in Pea
Assay for GSH-Conjugation of Benzo[c~]pyrene. Microsomal sus-
by vol, upper phase. The solvent system used for thin-layer chromatography on silica gel G (Frear and Swanson, 1973) was; (C) butanol-l/acetic acid/water, 12/3/5, by vol.
pension (50pl; containing 200-300pg protein), 0.2mM [7, 10-[14C]benzo[e]pyrene (50 pl; 1.9.103 d s- 1; dispersed in 0.3% (v/v) Triton X-100, 0.3% (v/v) ethyleneglycolmonomethylether), 125 mM GSH (5 gl).
Enzyme Preparation. Pea seeds were soaked overnight in tap water and were then germinated in the dark as described by Russell (1971). After 12 days, primary leaves and 10 cm epicotyl sections were collected separately and homogenized at 4 ~ C in 1 volume (per weight) of 50 mM Tricine x KOH, pH 7.5, by means of a household mixer (Krups model 3 Mix 3,000). The supernatant of a preliminary centrifugation (20 min, 10,000 g) was subjected to ultracentrifugation (90 min, 100,000g). The microsomal pellets were suspended in small volumes of 50 mM TricinexKOH, pH 7.5, and could be stored for several weeks at - 2 0 ~ C without loss of GSH S-cinnamoyl transferase activity. The supernatants from ultracentrifugation could also be stored for several weeks at - 2 0 ~ C. They were concentrated by ultrafiltration in an Amicon cell (UM-10 filter, 3.4.101 kN m -z, 5~ C) in order to remove most of an endogeneous transferase inhibitor. In most experiments (all of the kinetic experiments reported), the soluble enzymes were further purified by chromatography on a column of Sephacryl S200 (cf. Fig. 4).
Results and Discussion
Nature of Products Formed The GSH S-cinnamoyl adducts formed by the soluble and microsomal fractions of pea epicotyl and primary leaves (see below) were isolated by paper chromatography in solvent system (A). The previous chemical methods (Diesperger and Sandermann, 1978), in particular the release of dihydrocinnamic acid upon desulfurization by Raney nickel, indicated that in all cases GSH was linked to cinnamic acid by addition to the olefinic double bond, as shown in Fig. 1 A. The product formed from the herbicide fluorodifen depended on the pH-value of the incubation mixture (Fig. 2). At pH 9.0; a product with Rf-values of 0.5 [solvent system (A)] and 0.33 [solvent system (C)] was formed. This product was probably identical with the intact GSH-adduct of Figure 1 B (cf. Frear and Swanson, 1973). However, a faster migrating product with Rf-values of 0.7 [solvent system (A)] and 0.6 [solvent system (C)] was formed at pH 7.5. At an intermediate pH-value of 8.25, both products were detected (Fig. 2). These results appeared to indicate that the soluble enzyme fraction from pea seedlings contained some peptidase for a cleavage of the intact GSH-adduct of Fig. 1 B at pH-values below 9.0. No cleavage of the GSH S-cinnamoyl adduct of Fig. 1A could be observed in the same soluble enzyme fractions at pH 7.5.
Enzyme Assays General Procedure. All solutions used to determine enzyme activities contained 50 mM Tricine x KOH, pH 7.5. The standard incubations were carried out for 60 min at 30~ C and were terminated by the application of the incubation mixture to chromatography paper, followed by development in the solvent system (A). Control incubations with heat-denatured enzyme (5 rain, 95 ~ C) were always run in parallel, and the control values of radioactivity were subtracted from the values obtained with the non-denatured enzyme. The assay mixtures were prepared by mixing the following ingredients.
GSH S-Cinnamoyl TransferaseAssay. Enzyme solution (50 gl; containing 100 300 gg protein), 1.2 mM [3-14C]cinnamic acid (50 pl; 3.8.103 d s- 1), 50 mM Tricine x KOH (20 gl) or, where indicated, 6 mM NADPH (20 pl), 125 mM GSH (5 pl) or, where indicated, 75 mM 2-mercaptoethanol (5 gl). Assay for Cleavage of Fluorodifen (cf. Frear and Swanson, 1973). Enzyme solution (50 gl; containing appr. 100 pg protein), 176 mM [14C-CF3]fluorodifen (1 pl; 7.6.102 d s- 1), 6 mM GSH (50 pl).
{•
~OOH
COOH
GSH
CINNAMIC-ACI D
~)
GSH-ADDUCT
NO2 +
FLUORODINFE
GBH-ADDUC T
o@No2
Fig. 1A and B. Glutathione-dependent reactions catalyzed by enzymes from pea seedlings. A Conjugation with cinnamic acid. The chemical characterization of the GSH S-cinnamoyl adduct did not differentiate between a thioether linkage to either C2 or C3 of cinnamic acid. B Cleavage of the herbicide, fluorodifen (Frear and Swanson, 1973)
H. Diesperger and H. Sandermann, Jr. : Glutathione S-TransferaseActivities in Pea
645
71 Q
ORG IN I
A
I
FRONT
V L
aSH
I
I
Fig. 2. Reaction between [14 C-CFa]fluorodifen and glutathione as catalyzed by the purified soluble enzyme fraction from pea primary leaves. The figure shows the distributions of radioactivity on paper chromatograms [solvent system
?
0
A/
FRONT
(A)] of incubations performed at 1: pH 9.0, 2: pH 8.25 and 3: pH 7.5. Abbreviations: GSH = glutathione; F = fluorodifen
I
(]SH 1
o
i
....
1'o
I
2'o
F I
3'o
Previous studies with whole peanut plants have indicated that the intact GSH-adduct of Fig. 1 B was processed to the S-cysteinyl adduct 1 (Shimabukuro et al., 1978).
Kinetic Properties of the GSH S-Cinnamoyl Transferase Activities from Pea Seedlings The partially purified soluble enzymes and the microsomal activities had distinct pH-optima between pH 7.4 and 7.8, whereas, the non-enzymatic conjugation of GSH and cinnamic acid (cf. Diesperger and Sandermann, 1978; about 50 pmol adduct/min in the standard assay at pH 7.5) increased with increasing pH-values. The various transferase activities were linearly dependent on the amount of protein used (at least up to 300 gg protein per assay), as well as on the incubation time (up to 60 min). The purified, soluThe second split product of fluorodifen cleavage, p-nitrophenol (cf. Fig. 1 B), is further processed in peanut to the 6-O-malonyl-/L D-glucopyranosyl-derivative (Frear, 1976). A cell-free malonyltransfer from malonyl-SCoA to/LD-glucopyranosyl-p-nitrophenolate has recently been demonstrated with a highly purified malonyltransferase from parsley cell suspension cultures (U. Matern and
H. Sandermann, unpublished results)
,
40 CM
----
ble and microsomal transferase activities were abolished by heat-denaturation (5 min, 95 ~ C). In the case of the crude soluble extract from primary leaves, heating for 2 min at 95 ~ C resulted in a 2 8-fold stimulation, and transferase activity was only abolished after 20 min at 95 ~ C. Michaelis constants were determined by the Lineweaver-Burk procedure and were, in all cases, between 0.1-0.4 m M for cinnamic acid (in the presence of 5 m M GSH) and between 1-4 m M for GSH (in the presence of 0.5 m M cinnamic acid). V was 10 15nmol adduct/mg p r o t e i n x m i n for the purified soluble enzymes and 2 - 4 n m o l adduct/ mg protein x rain for the microsomal fractions. The microsomal transferase activity remained associated with the microsomal fraction after repeated washing and after centrifugation through a 5 cm cushion of 1 M glucose, 100 mM Tris-HC1, pH 7.5 (90 min, 100,000 g).
GSH S-Cinnamoyl Transfer in Other Plants The microsomal fraction from parsley cell suspension cultures (Diesperger and Sandermann, 1978) had V, 1 nmol adduct/mg protein x min. A microsomal frac-
646
H. Diesperger and H. Sandermann, Jr. : Gtutathione S-Transferase Activities in Pea
tion from potato slices (Rich and Lamb, 1977) had V, about 10 pmol adduct/mg protein • min. When tested in the presence of 1 mM NADPH, the potato microsomal fraction formed the Rf-0.4 GSH-adduct of cinnamic acid [solvent system (A)] which was probably derived from cinnamic acid 3.4-epoxide (Diesperger and Sandermann, 1978). In addition, smaller amounts of products which co-migrated with p-coumaric and caffeic acid [solvent systems (B)] and a cinnamoylcompound remaining at the origin in the solvent system (B), were formed. These various products were not formed by heat-denatured enzyme (5 rain, 95 ~ C) and required the presence of NADPH. Addition of the soluble protein fraction of potato slices led to the previously reported increase in p-coumaric acid formation (Rich and Lamb, 1977) at the expense of the cinnamoyl-compound at the origin.
No GSH S-cinnamoyl transferase activity was detected in the crude soluble enzyme fraction from potato slices (Rich and Lamb, 1977) or from parsley cell suspension cultures (Diesperger and Sandermann, 1978). It has not been determined whether this was caused by the presence of an endogeneous inhibitor which was present in the crude soluble extract from the pea seedlings (data not shown). Soybean cell suspension cultures were found to contain soluble and microsomal GSH S-cinnamoyl transferase activities with V, 2nmol adduct/mg proteinxmin for the microsomal fraction, and V, 4.5 nmol adduct/mg protein x min for the crude soluble fraction. Intact log-phase soybean cell suspension cultures (40 ml) were incubated for 2 h at 25 ~ C with [3-~4C]cinnamic acid (0.5 mM; 3.8.10 4 d s- 1) without the addition ofGSH. A 6% conversion to the GSH S-cinnamoyl adduct of Fig. 1A was found Upon paper chromatography in the solvent system (A), and about equal amounts of the adduct were isolated from the cells and from the growth medium. A 1.4% conversion was found in the autoclaved control cultures (cf. v. d. Trenck and Sandermann, 1978).
Alternative Substrates No detailed studies on alternative transferase substrates have been carried out. This section is, therefore, limited to some qualitative results obtained with the pea microsomal fractions. A GSH-adduct was formed when [3-14C]p-coumaric acid was employed instead of [3-14C]cinnamic acid, but [3-14C]dihydro cinnamic acid did not serve as a substrate. 2-Mercaptoethanol was able to partially replace GSH, and the adduct formed with cinnamic acid had an Rf-value of 0.25-0.4 in the solvent system (B). p-Coumaric acid has the same Rf-value in this solvent system which is in use for the assay of microsomal cinnamic acid 4-hydroxylase activity (Russell, 1971). Further-
more, 2-mercaptoethanol has been reported to be a strong activator of the pea cinnamic acid 4-hydroxylase (Russell, 1971). The present results indicate that the simple addition of 2-mercaptoethanol to cinnamic acid is also catalyzed by the pea microsomal fraction, and that this reaction may interfere with the chromatographic determination of p-coumaric acid. The microsomal fraction from pea primary leaves also catalyzed the formation of a GSH-adduct with the carcinogen benzo[~]pyrene, leading to a product with an Rf-value of 0.55 in the solvent system (A) [V, 30 pmol adduct/mg protein x h). This product was not formed in the absence of GSH or with heatdenatured enzyme (5 min, 95 ~ C). Subsequent results indicated that the microsomal fraction from pea primary leaves also catalyzes the formation of oxygenated metabolites of benzoMpyrene so that the GSHadduct may be formed as a secondary product (T. v. d. Trenck and H. Sandermann, unpublished resuits).
Inhibitors The following compounds gave less than 15% inhibition of microsomal GSH S-cinnamoyl transfer when added at 5 mM to the standard assay mixture; allyl alcohol, propachlor, styrene oxide, aldrin, dieldrin, endrin, DDE, DDMU, heptachlor, heptachlorepoxide, dihydrocinnamic acid, and atrazine. These inhibition effects were corrected for the appr. 25% inhibition given by the organic solvents used at final concentrations of 4%, v/v (methanol, dimethylsulfoxide or ethyleneglycol monomethylether). The addition of 5 mM styrene resulted in 27% inhibition; 5 mM cinnamyl alcohol inhibited by 76%, and 5 mM 3.4methylenedioxy-cinnamic acid gave complete inhibition. 2-Mercaptoethanol, which was accepted as an alternative substrate (see above), was a strong inhibitor of the soluble and microsomal GSH S-cinnamoyl transfer reactions (50% inhibition at 0 . 5 - 2 mM 2mercaptoethanol). Lineweaver-Burk plots (not shown) indicated a non-competitive type of inhibition.
Apparent Molecular Weights The soluble enzyme fractions were chromatographed on a column of Sephacryl S-200, and similar results were obtained for the extracts from epicotyl and primary leaves. In the experiment shown in Fig. 3, the soluble GSH S-cinnamoyl transferase activity appeared in two molecular weight regions corresponding to 37,000 and 150,000, respectively. In other experiments an additional peak corresponding to 75,000
H. Diesperger and H. Sandermann, Jr.: Glutathione S-Transferase Activities in Pea
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